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Abstract

Background

Malaria is an endemic disease in Yemen and is responsible for 4.9 deaths per 100,000
population per year and 43,000 disability adjusted life years lost. Although malaria
in Yemen is caused mainly by Plasmodium falciparum and Plasmodium vivax, there are no sequence data available on the two species. This study was conducted
to investigate the distribution of the Plasmodium species based on the molecular detection and to study the molecular phylogeny of these
parasites.

Methods

Blood samples from 511 febrile patients were collected and a partial region of the
18 s ribosomal RNA (18 s rRNA) gene was amplified using nested PCR. From the 86 positive
blood samples, 13 Plasmodium falciparum and 4 Plasmodium vivax were selected and underwent cloning and, subsequently, sequencing and the sequences
were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony
methods.

Results

Malaria was detected by PCR in 86 samples (16.8%). The majority of the single infections
were caused by P. falciparum (80.3%), followed by P. vivax (5.8%). Mixed infection rates of P. falciparum + P. vivax and P. falciparum + P. malariae were 11.6% and 2.3%, respectively. All P. falciparum isolates were grouped with the strain 3D7, while P. vivax isolates were grouped with the strain Salvador1. Phylogenetic trees based on 18 s
rRNA placed the P. falciparum isolates into three sub-clusters and P. vivax into one cluster. Sequence alignment analysis showed 5-14.8% SNP in the partial sequences
of the 18 s rRNA of P. falciparum.

Conclusions

Although P. falciparum is predominant, P. vivax, P. malariae and mixed infections are more prevalent than has been revealed by microscopy. This
overlooked distribution should be considered by malaria control strategy makers. The
genetic polymorphisms warrant further investigation.

Background

Malaria still continues to be a devastating global public health problem in more than
100 countries with 3.2 billion people being at risk [1]. Of this number, 300-500 million people contract the disease each year, resulting
in 2-3 million deaths [2]. This includes 1 million children of less than five years of age [3].

The genus Plasmodium consists of nearly 200 species that infect humans, birds, reptiles and mammals. It
belongs to the phylum Apicomplexa. Five Plasmodium species have been known to infect humans: P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi [4]. Much work on molecular phylogeny has focused on the relationship between Plasmodium species and the origin of human malaria [5-7]. Although the 18 s rRNA gene appears to be robust for phylogenetic analysis, its
sequences should be analysed carefully since the gene could be expressed as one of
the three types during the different developmental stages of the malaria parasite
[4,8]. Asexual type (A) and sporozoite type (S) were found in P. falciparum, P. berghei, P. vivax, and other species [4,9] while oocyst type (O) has been reported in P. vivax [10].

Yemen is a country in the East Mediterranean region where the highest incidence of
malaria has been registered after Afghanistan [11] and is responsible for 4.9 deaths per 100000 per year and 43000 disability adjusted
life years (DALYs) lost [12]. Giemsa microscopy is the most common diagnostic tool used in this country. This
technique has drawbacks, however, in terms of detecting low parasitemic cases and
the proper identification of mixed infection could actually underestimate the malaria
situation in Yemen. Thus, the molecular approach was applied for the first time, in
this country, to investigate the molecular epidemiology of malaria. The study will
indicate the magnitude of malaria co-infection which should be considered in clinical
case management. In addition, malaria strains, genetic polymorphism and the molecular
phylogeny of Yemen Plasmodium isolates will be studied.

Methods

Study area

The present study was conducted in five governorates with a total population of 7.9
million[13]. The selected governorates included Taiz and Hodeidah, which represent the mountainous
hinterland and coastal areas, respectively; and Rymah, Dhamar and Sana'a from the
highland areas. Most houses, especially in the rural areas, have a wooden roof. Occupations
include agriculture, fishing, livestock and handicrafts. The peak time of malaria
transmission in the coastal areas occurs in winter (October-April), while in the western
mountains, the peak occurs in the summer (May-September). In the highland areas, which
are located at more than 2000 metres above sea level, the transmission of malaria
occurs throughout the year [14]. Anopheles arabiensis is the main vector in the country but Anopheles culicifacies plays an important role in the transmission of malaria in the coastal area. Anopheles sergenti has also been reported in the mountainous hinterland and highland areas [14]. There is a paucity of comparative data on mortality and morbidity caused by malaria
in the three malaria endemic areas. An observational study showed that mortality rate
among children with severe malaria was 2.4% and 3.5% for costal and hinterlands, respectively
[15]. However, no data were available on mortality rate in the highland areas.

Sample collection and genomic DNA preparation

A total of 511 finger-prick blood samples were collected from symptomatic patients
attending hospitals or medical centres for the treatment of malaria. Blood from finger
pricks from febrile patients was collected and spotted on Whatman 3 MM filter papers
(Whatman International Ltd., Maidstone, England) and slides to prepare thin and thick
blood films. Each blood spot on Whatman filter paper was allowed to air-dry and was
stored in a separate sealed plastic bag at room temperature until DNA extraction was
required. Parasite genomic DNA was extracted from the blood collected on filter paper
using QIAgen DNA Mini Kit blood and tissue (QIAGEN, cat. no. 51306, Germany) according
to the manufacturer's instructions. Briefly, a disc was punched out from the blood
spot using a pre-flamed paper puncher and placed in 1.5 ml centrifuge tubes using
clean, flamed forceps.

Polymerase chain reaction (PCR)

Genus-and species-specific nested PCR assays based on the 18 s rRNA gene were used
to detect and identify Plasmodium species as previously reported [16]. Primary PCR was carried out using genus-specific primers (rPLU1 and rPLU5). PCR
reaction was run in a total of 25 μL reaction mixture containing 4 μL genomic DNA,
1× i-Taq™buffer including MgCl2 (iNtRON BIOTECHNOLOGY, Seoul, Korea), 250 μM dNTP (iNtRON BIOTECHNOLOGY, Seoul, Korea)),
200 nM of each primer and 1.25 U of i-Taq™DNA polymerase (iNtRON BIOTECHNOLOGY, Seoul,
Korea). Secondary PCR was carried out using species-specific primers (rFAL1/rFAL2,
rVIV1/rVIV2, rMAL1/rMAL2 and rOVA1/rOVA2); each primer pair was placed in a single
tube. PCR mixtures were as mentioned above, except that 2 μL of the first PCR product
was used as a template in the secondary PCR.

The cycling conditions for primary PCR were as follows: an initial denaturation step
at 95°C for 10 minutes, then 40 cycles at 94°C for 20 seconds, annealing at 55°C for
20 seconds, extension at 72°C for 1 minute, and a final extension at 72°C for 5 minutes.
The reaction was terminated by reducing the product temperature to 10°C. PCR products
were stored at -20°C until analysis. Secondary PCR used similar cycling conditions
except that the number of cycles was reduced to 35 cycles.

Cloning and DNA sequencing

The PCR product comprising 17 positive samples (13 P. falciparum and 4 P. vivax), representing different geographical areas, was selected for cloning and subsequent
sequencing. A partial sequence (~1200 bp) of the 18 s rRNA gene was amplified with
primers rPLU6 and rPLU5. PCR was carried out in a volume of 20 μl mixture containing
1× Phusion HF buffer (Finnzymes, Finland), 200 μM of each dNTP (Finnzymes) and 400
nM of each primer, and 1 U of Phusion DNA Polymerase (Finnzymes). The PCR conditions
were as follows: initial denaturation at 98°C for 30 seconds, followed by 40 cycles
of amplification at 98°C for 7 seconds, 59°C for 20 seconds and 72°C for 48 seconds
followed by a final extension step at 72 °C for 5 minutes. The amplified products
were cloned into Zero Blunt® vector according to the manufacturer's instructions (cat. No. K2700-20; Invitrogen,
USA). At least 20 colonies from each of transformation reactions were screened using
the rPLU6 and rPLU5 primers. Amplification was done in a 20 μl reaction mixture containing
1× reaction buffer (5× Green Go Taq Flexi Buffer, Promega Madison USA), 2 mM MgCl2 (Promega), 200 mM of each dNTP (Promega), 300 nM of each primer and 0.5 U Go Taq DNA
polymerase (Promega,). PCR conditions were as follows: initial denaturation of 94°C
for 10 minutes, followed by 30 cycles of amplification at 94°C at 45 seconds, annealing
at 55°C at 1 minute, extension at 72°C for 1 minute 30 seconds, followed by a final
extension step at 72°C at 10 minutes. Plasmids from clones having the correct insert
were extracted using QIAprep® Spin Miniprep kit (QIAgen, cat. no. 27106, Germany) following the manufacturer's instructions.
Purified plasmid containing the insert was sequenced in both directions using the
ABI PRISM® BigDye™ terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, USA) in an
3700 DNA Analyzer (Applied Biosystems, USA).

Phylogenetic analysis

DNA sequences (forward and reverse) were edited and the consensus sequence was created
using the BioEdit. Consensus sequences were multiple-aligned with previously published
sequences from the GenBank database using MEGA4 software http://www.megasoftware.netwebcite. Phylogenetic analysis was performed with the MEGA4 software. Two types of phylogenetic
analysis were used on the aligned sequences to assess relationships among isolates;
a distance-based Neighbor-Joining (NJ) analysis was performed calculated with the
Kimura 2-parameter [17], and Maximum Parsimony (MP) analysis was performed using the Close-Neighbour-Interchange
algorithm [18]. The reliability of the trees was assessed by the bootstrap method with 1,000 replications
[19]. Similarity searches were carried out using the Basic Local Alignment Search Tool
(BLAST) [20].

Results

A total of 511 blood samples from febrile patients were screened by using 18 s rRNA-based
nested PCR. Of the 511 samples, 86 (16.8%) were positive for malaria. A majority of
the malarial infections were due to P. falciparum (80.3%). Plasmodium vivax infections were seen in only 5.8% of the samples. Double infections with P. falciparum + P. vivax (11.6%) and P. falciparum + P. malariae (2.3%) were also detected (Table 1). The PCR product of 13 P. falciparum isolates and four of P. vivax were selected from different geographical areas including the coastal area, the hinterland
and the highland malaria endemic areas, and subjected to cloning and sequencing. The
17 sequences representing P. falciparum and P. vivax from this study (Table 2) and 13 sequences representing human, bird and primate malaria from the GenBank database,
were multiple aligned and analysed using the Neighbor-Joining (NJ) and Maximum Parsimony
(MP) methods.

To determine whether the genetic diversity is due to the heterogeneous types of Plasmodium 18 s rRNA or not, phylogenetic trees were re-constructed based on our sequences and
sequences representing different types of Plasmodium 18 s rRNA (A, O or S type) from the GenBank database. Both the NJ tree (Figure 2) and MP tree (Additional file 3) were consistent, placing all the Yemen isolates of P. falciparum except isolate R73 into one cluster and clade with P. falciparum type-S (GenBank Accession number M19173) (100 bootstrap). The isolate R73 was much closer to P. falciparum 18 s rRNA type-A (GenBank Accession number M19173) (100 bootstrap). All P. vivax isolates were grouped with P. vivax type-A in one cluster (GenBank Accession number U07367) (100 bootstrap).

Figure 2.Neighbor-Joining (NJ) tree, constructed based on the nucleotide sequences representing
the different types of 18 s rRNA (A, O or S type) from GeneBank and 13 sequences representing
13 P. falciparum isolates and 4 sequences representing 4 P. Vivax isolates from this study. Bootstrap support of more than 90% is indicated. Bold-type represents reference
sequences for Plasmodium species from GenBank.

Additional file 3.Maximum parsimony (MP) tree. The tree was constructed based on nucleotide sequences representing the different
types of 18 s rRNA (A, O or S type) from GeneBank and 13 sequences representing 13
P. falciparum isolates and 4 sequences representing 4 P. vivax isolates from this study. Bootstrap support of more than 90% is indicated. Bold-type
represents reference sequences for Plasmodium species from GenBank.

Discussion

This is the first study to apply molecular techniques to study the epidemiology and
phylogeny of malaria in Yemen. Malaria infection rate, using 18 s rRNA-based nested
PCR, was 16.8% which is comparable with previous studies carried out in Yemen based
on Giemsa microscopy [15,21-25]. PCR results showed that approximately 12% of the positive malaria cases were mixed
infection of P. falciparum and P. vivax or P. malariae which is a higher rate than has been hitherto reported. The high proportion of mixed
infection using molecular identification could be explained by the high sensitivity
of PCR [26] and the low level parasitaemia of the dominated species which is under the diagnostic
threshold of Giemsa microscopy. Accurate identification of mixed infection with malaria
species is very important for proper clinical case management. Malaria treatment policy
differs depending on the infecting species. Thus, in the case of mixed infection,
if only one species is treated, the other may establish a new episode of malaria.

Plasmodium falciparum is the dominant malaria species in Yemen followed by P. vivax, whereas P. malariae has rarely been reported in Yemen. The current study highlighted the possibility that
P. malariae is distributed more than expected and it is often overlooked due to low levels of
parasitaemia and the shortcoming of microscopy, the only technique used for malaria
detection and identification in the country. Furthermore, P. malariae may be obscured by the dominant species. The underestimation of P. malariae distribution has been reported in other countries [27-30]

Phylogenetic analysis using the NJ method grouped Yemen isolates of P. falciparum into one main cluster. Within the main cluster, the Yemen isolates were placed in
three sub-clusters (Figure 1). These clusters were supported by cladograms using the MP method. One sub-cluster
contained 8 isolates and P. falciparum 3D7 (GenBank Accession number AE014186) [31]. The second sub-cluster included four isolates and P. falciparum (GenBank Accession number AF145334) which was isolated from Papua New Guinea [32]. The isolate R73 formed a separate cluster with P. falciparum (GenBank Accession number AL844506). Phylogenetic analysis based on our sequences and sequences from the GenBank representing
the secondary structure of 18 s rRNA showed that isolate R73 that diverged in a separate
cluster may be A-type ribosomal rRNA, while the other two sub-clusters are S-type
rRNA. Thus, genetic diversity within the Yemen isolates that grouped in one cluster
with the S-type rRNA may be not due to the heterogeneity of 18 s rRNA genes. Genetic
diversity may provide a mechanism for drug resistance which may affect any intervention
strategy based on treatment. Furthermore, PCR-based specific diagnosis of Plasmodium species may fail due to sequence variations in the priming regions, thus leading to
false negative results. The low sensitivity of PCR-based diagnosis due to genetic
mutations has been previously reported [30].

Conclusions

In conclusion, malaria is a major public health problem in Yemen. Although P. falciparum is predominant, P. vivax, P. malariae and mixed infections are more prevalent than has been revealed by microscopy. This
overlooked distribution should be considered by malaria control strategy makers. Sequences
from P. falciparum isolates showed high genetic polymorphisms that may not be related to the variants
of ribosomal RNA expressed in the different stages of malaria parasites which warrant
further studies.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

AMQA, MAKM, FMY and AAA and designed the study; AMQA dealt with study subjects in
the field, carried out the laboratory work and collected the data; AMQA and MAKM performed
the statistical analysis; AMQA, MAKM, FMY and AAA and interpreted the data; AMQA and
MAKM drafted the manuscript; AMQA, FMY, AAA, MAKM contributed to the revision of the
manuscript. All authors read and approved the final manuscript. AMQA and FMY are guarantors
of the paper.

Acknowledgements

The authors thank all the technical staff in the hospitals and medical centres in
the five governorates, especially Sultan Ayesh, Fawaz Al-Soroorey, Ashraf Saleh and
Ahlam Al-Kobati. Thanks are also due to Entesar Mansour M.H and Nemah O. M. Bin Shuaib
for their assistance in the laboratory work. The authors also thank Dr. Hesham M.
Al-Mekhlafi for his assistance. The study was funded by a research grant from the
University of Malaya, Kuala Lumpur, Malaysia (Research Code PS175/2008C).

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